Recombinant proteins are an emerging class of therapeutic agents. Such recombinant therapeutics have engendered advances in protein formulation and chemical modification. Such modifications can protect therapeutic proteins, primarily by blocking their exposure to proteolytic enzymes. Protein modifications may also increase the therapeutic protein""s stability, circulation time, and biological activity. A review article describing protein modification and fusion proteins is Francis (1992), Focus on Growth Factors 3:4-10 (Mediscript, London), which is hereby incorporated by reference.
One useful modification is combination with the xe2x80x9cFcxe2x80x9d domain of an antibody. Antibodies comprise two functionally independent parts, a variable domain known as xe2x80x9cFabxe2x80x9d, which binds antigen, and another domain known as xe2x80x9cFcxe2x80x9d, which links to such effector functions as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas an Fab is short-lived. Capon et al. (1989), Nature 337:525-31. When constructed together with a therapeutic protein, an Fc domain can provide longer half-life or incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. Id. Table 1 summarizes use of Fc fusions known in the art.
Despite their advantages, use of Fc fusion molecules may be limited by misfolding upon expression in a desired cell line. Such misfolded Fc fusion molecules may generate an immune response in vivo or may cause aggregation or stability problems in production.
The present invention concerns a process by which a misfold in an Fc fusion molecule can be prevented or corrected. In one embodiment, the process comprises:
(a) preparing a fusion molecule comprising (i) a pharmacologically active domain and (ii) an Fc domain;
(b) treating the fusion molecule with a copper (II) halide; and
(c) isolating the treated fusion molecule.
The preferred copper (II) halide is CuCl2. The preferred concentration thereof is at least about 10 mM for fusion molecules prepared in E. Coli; at least about 30 mM for fusion molecules prepared in CHO cells.
An alternative embodiment of the process comprises the following steps:
(a) preparing a fusion molecule comprising (i) a pharmacologically active domain and (ii) an Fc domain;
(b) treating the fusion molecule with guanidine HCl at a concentration of at least about 4 M;
(c) increasing the pH to about 8.5; and
(d) isolating the treated fusion molecule.
Each of these processes can be employed with any number of pharmacologically active domains. Preferred pharmacologically active domains include OPG proteins, leptin proteins, TNF-xcex1 inhibitors (e.g., wherein the fusion molecule is etanercept), IL-1 inhibitors (e.g., IL-1ra proteins, which are preferred), and TPO-mimetic peptides. Also within the claimed process are molecules in which the pharmacologically active compound is an antibody. The Fc domain preferably has a human sequence, with an Fc sequence derived from IgG1 most preferred. An exemplary Fc sequence is shown in FIG. 5 hereinafter.
Although mostly contemplated as therapeutic agents, compounds of this invention may also be useful in screening for such agents. For example, one could use an Fc-peptide (e.g., Fc-SH2 domain peptide) in an assay employing anti-Fc coated plates. The vehicle, especially Fc, may make insoluble peptides soluble and thus useful in a number of assays.
The compounds prepared by the process of this invention may be used for therapeutic or prophylactic purposes by formulating them with appropriate pharmaceutical carrier materials and administering an effective amount to a patient, such as a human (or other manual) in need thereof.
Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.